Infantile respiratory distress syndrome (RDS) is a leading cause of death in the first 28 days of life. It strikes 1 in 100 babies worldwide and about 10 percent die. The syndrome rarely occurs in term infants but is generally associated with immaturity and in low-birth weight (under 2 kg). Adult RDS shows similar clinical characteristics and pathophysiology as the infantile disease and is managed in the intensive care facility in a similar fashion. The adult disease has diverse etiologies and results from lung insults such as diffuse infections, aspiration of the gastric contents or water, inhalation of irritants andtoxins and pulmonary edema arising from such sources as narcotic overdose. RDS is correlated with an absence or dysfunction of the lung surfactant which coats the alveoli of the lungs where gas exchange occurs.
Lung surfactant is composed primarily of lipid (90%) and contains a minor component of various proteins (10%). The primary lipid type is phospholipid which constitutes 97% of the lipid. 80% of this phospholipid is phosphatidylcholine (PC) and 10% is phosphatidyglycerol (PG). 70-80% of the phospholipid acyl chains are saturated and 85-90% of these are 16-carbons in length. The major lipid is dipalmitoylphosphatidylcholine (DPPC). The protein component of lung surfactant is also heterogeneous but more poorly characterized. The major protein is .about.32 kd and its sequence has been predicted from its cDNA (White et al. (1985) Nature 317, 361-363) and it has been cloned (Floros, et al. Journal of Biological Chemistry (1985) 260, 495-500). Minor proteins of lower molecular weight have also been isolated and several sequences have been reported (Warr, et al. (1987) Proceedings of the National Academy of Sciences USA 82, 7915-7919; Glasser, et al. (1988) Journal of Biological Chemistry 263, 9; Johansson, et al. (1988) FEBS Letters. 232, 61-64; Glasser, et al. (1987) Proceedings of the National Academy of Sciences USA 84, 4007; Revak, et al. (1988) Journal of Clinical Investigation 81, 826-833).
Infantile RDS has been treated with animal lung lavages (Smyth, et al. (1983) Pediatrics 71, 913-917; McCord, et al. (1988) Arch. Dis. Child. 63, 10-16) and human lung surfactant obtained from human amniotic fluid (Hallman, et al. (1983) Pediatrics 71, 473-482; (1985) J. Pediatr. 106, 963-969; Merritt, et al. (1986) N. Enql. J. Med. 13, 785-790) with considerable success. Cow lung lavages extracted with organic solvents and mixed with synthetic lipids are also effective (Fujiwara, et al. (1980) Lancet 1, 55-59; Noach, et al. (1986) Eur. J. Respir. Dis. 69, 321-335; Kwong, et al. (1985) Pediatrics 76, 585-592; Enhorning, et al., (1985) Pediatrics 76, 145-153; Shapiro, et al. (1985) Pediatrics 76, 593-599; Gitlin, et al. (1987) Pediatrics 79, 31-37; Raju, et al. (1987) Lancet 1, 651-656; Halliday, et al (1984) Lancet 1, 476-478). However, pure synthetic mixtures have not consistently been shown to be active (Morley, et al., (1981) Lancet 1, 64-68; Ten Centre Study Group (1987) Br. Med. J. 294, 991-996; Milner, et al. (1983) Arch. Dis. Child. 58, 458-460; Milner, et al. (1983, ibid 59, 369-371; and Wilkinson, et al. (1985) Lancet 2, 287-291). Only a few clinical studies using surfactants in adult RDS are known, i.e., (Lochman, et al., (1988) Adv. Exp. Med. Biol. 222, 511-517), however, the potential value of such therapy has been indicated in the literature (van Golde, et al. Physiol. Rev. 68, 374-455).
Although limited success with pure lipid mixtures has been reported, it appears that a protein component of lung surfactant dramatically improves activity, such as is observed with natural lung surfactants (Hall. et al., Am. Rev. Rspir. Dis. (1992) 145, 24-30). One approach is to use genetically engineered proteins with sequences based on the amino acid sequences predicted from the cDNA of the lung surfactant proteins. An alternative method, which is the subject of the present invention is to synthesize peptides with physical properties suited to the function of lung surfactant when in combination with defined lipid components, but with sequences unrelated to the sequences of proteins isolated from lung surfactant.
The present invention is concerned with short peptides of 3 to 4 amino acids having an N-acyl group and compositions comprising a mixture of synthetic polypeptides and lipids effective in treating RDS. The synthetic polypeptides have unique sequences which are not found in the sequences of the mammalian proteins isolated from lung lavages. The polypeptides may be used singly in mixtures with lipid or in combination in mixtures of lipid. The polypeptide comprises a minor component of the surfactant mixture.
Recently it was demonstrated that of the several classes of lipid-binding peptides, having mixtures of an amphipathic .alpha.-helical peptide with about 10-24 amino acids, with dipalmitoylphosphatidylcholine (DPPC) were effective in restoring quasi-static lung compliance in vitro and gas exchange In vivo in lavaged lungs. The present invention has significantly decreased the size of the peptide required to only 3 to 4 amino acids by modifications of the amino and carboxy terminal.
A helical wheel representation of an amphipathic .alpha.-helical ten-residue peptide (for description of the amphipathic .alpha.-helical peptide see McLean, L. R. et al. Biochem., 1991, 30, 31) is used to develop a model for three and four residue peptides. When looking down the barrel of the .alpha.-helix, the side chains of the residues indicate a hydrophobic face and a hydrophilic face on the helix. A four residue peptide represents a single turn of this .alpha.-helix with the required hydrophobic and hydrophilic face present. A three residue peptide represents a contricted turn of the .alpha.-helix with the hydrophobic and hydrophilic face still present.
The chemical composition of the present invention is novel with regard to the short peptide component of 3-4 amino acids adjoined to an acyl or hydrophobic sequence of 8 to 20 carbons and the combination of such peptides with lipids in a synthetic lung surfactant which is medicinally effective in the treatment of respiratory distress syndromes. The composition of the present invention may be prepared in high purity and in a standardized fashion as it is a defined mixture of synthetic components. Also, the components are not derived from animal sources which minimizes the risk of contamination by viruses and bacteria.